Mocetinostat activates Krüppel-like factor 4 and protects against tissue destruction and inflammation in osteoarthritis

Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat — a class I selective histone deacetylase (HDAC) inhibitor — had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.

Supplemental Figure 4. Regulation of anabolic and catabolic genes by mocetinostat in human meniscal cells.(A, B) Cells were treated with 10 µM of mocetinostat or DMSO, and RNA was collected 24 hours after initiation of treatment.mRNA levels are expressed as means±SE, relative to DMSO (n=6 donors).*P<0.05,**P<0.01,paired t-test.
Frey test in mocetinostat-treated mice after DMM surgery.(A, B) Related to the experiments in Figure 5, results of von Frey test preoperatively (A) and at 5 weeks postoperatively (B) in mocetinostat-treated mice after DMM surgery are shown.Numbers of paw withdrawal from 5 stimulations per filament per mouse are expressed as means±SE.n=14 for DMM + 10 mg/kg of mocetinostat, and n=15 for the other groups.*P<0.05,**P<0.01,Dunn's test versus DMM + vehicle.NS, not significant.Results of Kruskal-Wallis test are shown in Supplemental Table17.
scores in mocetinostattreated mice after DMM surgery.This figure shows data related to the experiments in Figure 5. n=14 for DMM + 10 mg/kg of mocetinostat, and n=15 for the other groups.*P<0.05,**P<0.01,Dunn's test versus DMM + vehicle.All quantitative data are expressed as means±SE, and results of Kruskal-Wallis test are shown in Supplemental Table17.
mice after DMM surgery.This figure shows data related to the experiments in Figure 5. (A, B) Immunohistochemistry for KLF4 in knee cartilage was performed.n=14 for DMM + 10 mg/kg of mocetinostat, and n=15 for the other groups.Scale bars, 60 µm.*P<0.05,**P<0.01,Dunn's test versus DMM + vehicle.All quantitative data are expressed as means±SE, and results of Kruskal-Wallis test are shown in Supplemental Table 17.
mice after DMM surgery.This figure shows data related to the experiments in Figure 5. (A, B) Immunohistochemistry for ADAMTS5 in knee cartilage was performed.n=13 for Sham + vehicle, n=14 for DMM + vehicle, n=15 for DMM + 2 mg/kg of mocetinostat, and n=14 for DMM + 10 mg/kg of mocetinostat.Scale bars, 60 µm.*P<0.05,**P<0.01,Dunn's test versus DMM + vehicle.All quantitative data are expressed as means±SE, and results of Kruskal-Wallis test are shown in Supplemental Table 17.
mice after DMM surgery.This figure shows data related to the experiments in Figure 5. (A, B) mice after DMM surgery.This figure shows data related to the experiments in Figure 5. (A, B) Immunohistochemistry for MMP13 in knee cartilage was performed.n=13 for Sham + vehicle, n=14 for DMM + vehicle, n=15 for DMM + 2 mg/kg of mocetinostat, and n=14 for DMM + 10 mg/kg of mocetinostat.Scale bars, 60 µm.*P<0.05,**P<0.01,Dunn's test versus DMM + vehicle.All quantitative data are expressed as means±SE, and results of Kruskal-Wallis test are shown in Supplemental Table 17.mice after DMM surgery.This figure shows data related to the experiments in Figure 5. (A, B) Immunohistochemistry for FOXO1 in knee cartilage was performed.n=13 for Sham + vehicle, n=14 for DMM + vehicle, n=15 for DMM + 2 mg/kg of mocetinostat, and n=14 for DMM + 10 mg/kg of mocetinostat.Scale bars, 60 µm.*P<0.05,**P<0.01,Dunn's test versus DMM + vehicle.All quantitative data are expressed as means±SE, and results of Kruskal-Wallis test are shown in Supplemental Table 17.Cells were transfected with siRNAs, and were treated with 2 µM mocetinostat or DMSO.Total protein was collected 24 hours after initiation of treatment.(A) Representative blots from three independent experiments.(B) Normalized intensities of protein bands are expressed as means±SE, relative to DMSO + control (n=3 independent experiments).*P<0.05,**P<0.01,Sidak's multiple comparison test.Results of two-way mixed-effects ANOVA test are shown in Supplemental

Numbers of paw withdrawal
of Kruskal-Wallis test are shown in Supplemental Table 17.

Table 3 . Cell survival rates of SW1353 cells treated with the 18 identified compounds.
Cells were treated with either of the identified compounds or DMSO for 24 hours.Each compound was used with different doses (20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 µM, 2 µM, 5 µM, 10 µM, 20 µM and 30 µM), and cell survival rates of each compound were measured at the highest doses with normal cell viability (microscopically observed).Cell survival rates are expressed as means±SE (n=4 from four independent experiments).Dunn's test, versus DMSO.Results of Kruskal-Wallis test are shown in Supplemental Table17.

Table 4 . Summary of experiments for SW1353 cells treated with the compounds upregulating KLF4.
The results of statistical analyses are summarized for the experiments shown in Figure1B.*and**, significant upregulation with P<0.05 and P<0.01, respectively.DR, significant downregulation.NS, non-significant changes.Dunnett's test versus DMSO (n=4 from four independent experiments).Results of oneway mixed-effects ANOVA test are shown in Supplemental Table17.

Supplemental Table 13. Summary of enriched pathways common between URGs of RNA-seq and URPs of TMT-MS.
Enriched pathways common between URGs of RNA-seq and URPs of TMT-MS